Antibody-based therapeutics have now had success in the clinic for the treatment and diagnosis of a variety of cancers. The affinity and specificityof the antibody for the target ligand determines the specificity of therapeutic delivery and off-targe side effects. The discovery and optimization of high affinity antibodies to important cancer targets is critical for the development of new clinical approaches but is still a very challenging task. A robust, eukaryotic technology that is comparable to phage and yeast display would overcome the protein translation limitations of microorganisms and thereby improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression sys- tem for clinical production. Our long-term goal is to improve the targeting of cancer therapeutics by increasing delivery specificity and distribution in the target tissue thereby improving efficacy while reducing/eliminating off-target side effects. The overall objective of this application is to validate the utility and efficiency o a novel antibody display platform based on the eukaryotic retrovirus, Avian Leukosis Virus (ALV), to affinity optimize anti- bodies in eukaryotic cells, and to advance the development of the technology for initial antibody discovery and improved library screening. Our central hypothesis is: the characteristics of ALV replication and ALV polypeptide display offer a robust, eukaryotic version of bacteriophage display, thereby enabling an improvement in the diversity of properly processed and functional scFv variants that can be screened and affinity optimized to improve promising antibody candidates compared to antibody display and affinity maturation using microorganisms. We plan to test our central hypothesis and, thereby, attain the objective of the application by pursuing the following specific aims: Aim 1: Validate the utility of ALV virus display technology for antibody affinity optimization. Aim 2: Advance the development of the ALV antibody display platform. Aim 3: Evaluate the specificity, cytotoxicity, and biodistribution o affinity-matured antibody fragments in relevant cancer models. We will affinity mature a series of lead scFvs and single-domains that recognize two newly emerging cancer targets, glypican-3 (liver cancers) and mesothelin (mesothelioma, ovarian cancer) and demonstrate superior cancer cell killing with targeting using the optimized antibody fragments. The rationale for the proposed experiments is to solve the need for a robust eukaryotic polypeptide display platform for the discovery and affinity maturation of antibodies, offering a powerful addition to the existig display technologies that use microorganisms. The significance of our contribution here is supplying just such a technology. The research proposed in this application is innovative because it represents a new and substantive departure from the status quo of antibody display using platforms based on microorganisms, providing a significant improvement and extension of mammalian cell display by offering both virus and cell display platforms in a eukaryotic expression system.